Global Analysis of Protein Stability by Temperature and Chemical Denaturation
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Protein Unfolding (Model Peptide Helix/Helix-Coil Transition/Denaturant Effects) J
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 185-189, January 1995 Biochemistry Urea unfolding of peptide helices as a model for interpreting protein unfolding (model peptide helix/helix-coil transition/denaturant effects) J. MARTIN SCHOLTZ*tt, DOUG BARRICK*§, EUNICE J. YORK0, JOHN M. STEWART$, AND ROBERT L. BALDWIN*: *Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 97305-5307; tDepartment of Medical Biochemistry and Genetics, Center for Macromolecular Design, Texas A&M University, College Station, TX 77843-1114; and 1Department of Biochemistry, University of Colorado Health Sciences Center, Denver, CO 80262 Contributed by Robert L. Baldwin, August 29, 1994 ABSTRACT To provide a model system for understand- The peptides examined here consist primarily of alanine;"1 ing how the unfolding of protein a-helices by urea contributes thus side-chain interactions are minimal, and the primary to protein denaturation, urea unfolding was measured for a structural and thermodynamic changes during helix unfolding homologous series of helical peptides with the repeating should result from the peptide backbone. Because these helical sequence Ala-Glu-Ala-Ala-Lys-Ala and chain lengths varying peptides lack hydrophobic cores and long-range tertiary in- from 14 to 50 residues. The dependence of the helix propa- teractions, the thermodynamics of solvent denaturation of gation parameter of the Zimm-Bragg model for helix-coil secondary structure can be measured directly. Although sec- transition theory (s) on urea molarity ([urea]) was deter- ondary structure formation must be an integral part of the mined at 0°C with data for the entire set of peptides, and a protein folding reaction, these other factors (hydrophobic core linear dependence ofIns on [urea] was found. -
State Folding of Globular Proteins
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 February 2020 doi:10.20944/preprints202002.0290.v1 Peer-reviewed version available at Biomolecules 2020, 10, 407; doi:10.3390/biom10030407 Review The Molten Globule, and Two-State vs. Non-Two- State Folding of Globular Proteins Kunihiro Kuwajima,* Department of Physics, School of Science, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and School of Computational Sciences, Korea Institute for Advanced Study (KIAS), Seoul 02455, Korea ; [email protected] * Correspondence: [email protected] Abstract: From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate. -
Acid Dissociation Constant - Wikipedia, the Free Encyclopedia Page 1
Acid dissociation constant - Wikipedia, the free encyclopedia Page 1 Help us provide free content to the world by donating today ! Acid dissociation constant From Wikipedia, the free encyclopedia An acid dissociation constant (aka acidity constant, acid-ionization constant) is an equilibrium constant for the dissociation of an acid. It is denoted by Ka. For an equilibrium between a generic acid, HA, and − its conjugate base, A , The weak acid acetic acid donates a proton to water in an equilibrium reaction to give the acetate ion and − + HA A + H the hydronium ion. Key: Hydrogen is white, oxygen is red, carbon is gray. Lines are chemical bonds. K is defined, subject to certain conditions, as a where [HA], [A−] and [H+] are equilibrium concentrations of the reactants. The term acid dissociation constant is also used for pKa, which is equal to −log 10 Ka. The term pKb is used in relation to bases, though pKb has faded from modern use due to the easy relationship available between the strength of an acid and the strength of its conjugate base. Though discussions of this topic typically assume water as the solvent, particularly at introductory levels, the Brønsted–Lowry acid-base theory is versatile enough that acidic behavior can now be characterized even in non-aqueous solutions. The value of pK indicates the strength of an acid: the larger the value the weaker the acid. In aqueous a solution, simple acids are partially dissociated to an appreciable extent in in the pH range pK ± 2. The a actual extent of the dissociation can be calculated if the acid concentration and pH are known. -
Kinetic Folding Mechanism of Erythropoietin
Biophysical Journal Volume 96 May 2009 4221–4230 4221 Kinetic Folding Mechanism of Erythropoietin Douglas D. Banks,* Joanna L. Scavezze, and Christine C. Siska Department of Analytical and Formulation Sciences, Amgen, Seattle, Washington 98119-3105 ABSTRACT This report describes what to our knowledge is the first kinetic folding studies of erythropoietin, a glycosylated four-helical bundle cytokine responsible for the regulation of red blood cell production. Kinetic responses for folding and unfolding reactions initiated by manual mixing were monitored by far-ultraviolet circular dichroism and fluorescence spectroscopy, and folding reactions initiated by stopped-flow mixing were monitored by fluorescence. The urea concentration dependence of the observed kinetics were best described by a three-state model with a transiently populated intermediate species that is on-pathway and obligatory. This folding scheme was further supported by the excellent agreement between the free energy of unfolding and m-value calculated from the microscopic rate constants derived from this model and these parameters determined from separate equilibrium unfolding experiments. Compared to the kinetics of other members of the four-helical bundle cytokine family, erythro- poietin folding and unfolding reactions were slower and less susceptible to aggregation. We tentatively attribute these slower rates and protection from association events to the large amount of carbohydrate attached to erythropoietin at four sites. INTRODUCTION Erythropoietin (EPO) is a 30 kD hormone produced EPO is an attractive protein-folding model due to its inter- primarily in the kidneys and is responsible for regulating mediate size and high degree of folding reversibility. A red blood cell production by stimulating late erythroid wealth of information has already been learned studying precursor cells (1). -
Protein Folding: New Methods Unveil Rate-Limiting Structures
UNIVERSITY OF CHICAGO PROTEIN FOLDING: NEW METHODS UNVEIL RATE-LIMITING STRUCTURES A DISSERTATION SUBMITTED TO THE FACULTY OF THE DIVISION OF THE BIOLOGICAL SCIENCES AND THE PRITZKER SCHOOL OF MEDICINE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY BY BRYAN ANDREW KRANTZ CHICAGO, ILLINOIS AUGUST 2002 Copyright © 2002 by Bryan Andrew Krantz All rights reserved ii TO MY WIFE, KRIS, AND MY FAMILY iii Table of Contents Page List of Figures viii List of Tables x List of Abbreviations xi Acknowledgements xii Abstract xiii Chapter 1.0 Introduction to Protein Folding 1 1.1 Beginnings: Anfinsen and Levinthal 1 1.1.1 Intermediates populated by proline isomerization 2 1.1.2 Intermediates populated by disulfide bond formation 4 1.1.3 H/D labeling folding intermediates 7 1.1.4 Kinetically two-state folding 8 1.2 Present thesis within historical context 11 1.3 The time scales 12 1.3.1 From microseconds to decades 12 1.3.2 A diffusive process 13 1.3.3 Misfolding and chaperones 13 1.4 The energies 14 1.4.1 The hydrophobic effect 14 1.4.2 Conformational entropy 15 1.4.3 Hydrogen bonds 15 1.4.4 Electrostatics 16 1.4.5 Van der Waals 19 1.5 Surface burial 19 1.6 Modeling protein folding 20 1.6.1 Diffusion-collision and hydrophobic collapse 21 1.6.2 Search nucleation 22 1.6.3 Are there intermediates? 23 1.6.4 Pathways or landscapes? 27 2.0 The Initial Barrier Hypothesis in Protein Folding 30 2.1 Abstract 30 2.2 Introduction 30 2.3 Early intermediates 41 2.3.1 Cytochrome c 41 2.3.2 Barnase 46 2.3.3 Ubiquitin -
The Inverted Chevron Plot Measured by NMR Relaxation Reveals a Native-Like Unfolding Intermediate in Acyl-Coa Binding Protein
The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein Kaare Teilum*, Flemming M. Poulsen†, and Mikael Akke*‡ *Department of Biophysical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden; and †Institute of Molecular Biology and Physiology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen, Denmark Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved March 14, 2006 (received for review October 18, 2005) The folding kinetics of bovine acyl-CoA binding protein was stud- equilibrium after rapid perturbation of the system. For example, ied by 15N relaxation dispersion measurements under equilibrium the response to a rapid change in denaturant concentration can conditions. Relaxation dispersion profiles were measured at sev- be monitored by using stopped-flow methods. In this fashion, eral concentrations of guanidine hydrochloride (GuHCl). The un- folding (unfolding) rates can be measured sensitively only at folding rate constant (ku) was determined under conditions favor- denaturant concentrations below (above) the midpoint of the ing folding, for which the folding rate constant (kf) dominates the folding transition, where relaxation to the new equilibrium is relaxation in stopped-flow kinetic measurements. Conversely, kf dominated by the folding (unfolding) rate constant. Hence, long was determined under conditions favoring unfolding, for which ku extrapolations are usually necessary to extract the unfolding dominates stopped-flow data. The rates determined by NMR there- rates under physiological conditions at zero denaturant concen- fore complement those from stopped-flow kinetics and define an tration. Protein unfolding under conditions favoring the native ‘‘inverted chevron’’ plot. -
Identification and Characterization of Protein Folding Intermediates Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli
Identification and characterization of protein folding intermediates Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli To cite this version: Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli. Identifica- tion and characterization of protein folding intermediates. Biophysical Chemistry, Elsevier, 2007, 128 (2-3), pp.105. 10.1016/j.bpc.2007.04.008. hal-00501662 HAL Id: hal-00501662 https://hal.archives-ouvertes.fr/hal-00501662 Submitted on 12 Jul 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ÔØ ÅÒÙ×Ö ÔØ Identification and characterization of protein folding intermediates Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini- Allocatelli PII: S0301-4622(07)00087-7 DOI: doi: 10.1016/j.bpc.2007.04.008 Reference: BIOCHE 4952 To appear in: Biophysical Chemistry Received date: 29 March 2007 Revised date: 16 April 2007 Accepted date: 16 April 2007 Please cite this article as: Stefano Gianni, Ylva Ivarsson, Per Jemth, Maurizio Brunori, Carlo Travaglini-Allocatelli, Identification and characterization of protein folding inter- mediates, Biophysical Chemistry (2007), doi: 10.1016/j.bpc.2007.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. -
Revisiting Absorbance at 230Nm As a Protein Unfolding Probe
Analytical Biochemistry 389 (2009) 165–170 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Revisiting absorbance at 230 nm as a protein unfolding probe Pei-Fen Liu a,b, Larisa V. Avramova c, Chiwook Park a,b,c,* a Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Heine Pharmacy Building, Room 224A, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA b Purdue University Interdisciplinary Life Science Program (PULSe), Purdue University, West Lafayette, IN 47907-2091, USA c Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907-2057, USA article info abstract Article history: Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring pro- Received 19 February 2009 tein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absor- Available online 24 March 2009 bance at 230 nm (A230) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A230 as a structural Keywords: probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli Protein stability maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored UV absorbance their unfolding in urea and guanidinium chloride with A . Significant changes in A were observed 230 nm 230 230 with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully Conformational change Unfolding kinetics determined by measuring the change in A230 in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A230 under denaturing conditions. -
Kinetic Evidence for a Two-Stage Mechanism of Protein Denaturation by Guanidinium Chloride
Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride Santosh Kumar Jhaa,1 and Susan Marquseea,b,2 aCalifornia Institute for Quantitative Biosciences and bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220 Edited by Robert L. Baldwin, Stanford University, Stanford, CA, and approved February 14, 2014 (received for review August 14, 2013) Dry molten globular (DMG) intermediates, an expanded form of hydrophobic core, resulting in global structural disruption. Ex- the native protein with a dry core, have been observed during perimental support for this model has, however, been scarce and denaturant-induced unfolding of many proteins. These observa- indirect (4, 6, 13, 26). tions are counterintuitive because traditional models of chemical Here, we demonstrate that the well-studied protein Escherichia denaturation rely on changes in solvent-accessible surface area, coli ribonuclease HI (RNase H) (28–30) also rapidly populates and there is no notable change in solvent-accessible surface area a DMG state when exposed to denaturant and use this observation during the formation of the DMG. Here we show, using multisite to explore the denaturant dependence of the DMG. We use fluorescence resonance energy transfer, far-UV CD, and kinetic multisite fluorescence resonance energy transfer (FRET) and thiol-labeling experiments, that the guanidinium chloride (GdmCl)- kinetic thiol-labeling measurements to dissect the temporal or- induced unfolding of RNase H also begins with the formation of der of protein expansion, side-chain disruption, and water sol- the DMG. Population of the DMG occurs within the 5-ms dead time vation during GdmCl-induced unfolding. -
I. Equilibrium Unfolding Studies of Cytochrome C. II. the Role of Helix 1 Aspartates in the Stability and Conversion of Prion Protein
University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2003 I. Equilibrium unfolding studies of cytochrome C. II. The role of helix 1 aspartates in the stability and conversion of prion protein Jonathan O'Connor Speare The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Speare, Jonathan O'Connor, "I. Equilibrium unfolding studies of cytochrome C. II. The role of helix 1 aspartates in the stability and conversion of prion protein" (2003). Graduate Student Theses, Dissertations, & Professional Papers. 9470. https://scholarworks.umt.edu/etd/9470 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. Maureen and Mike MANSFIELD LIBRARY The University of Montana Permission is granted by the author to reproduce this material in its entirety, provided that this material is used for scholarly purposes and is properly cited in published works and reports. **Please check "Yes" or "No" and provide signature** Yes, I grant permission ___ No, I do not grant permission ___ Author's Signature:__ Date: ____ 3 Any copying for commercial purposes or financial gain may be undertaken only with the author's explicit consent. 8/98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -
Microcalorimetric Techniques
Microcalorimetric techniques Isothermal titration calorimetry (ITC) Differential scanning calorimetry (DSC) Filip Šupljika [email protected] Laboratory for the study of interactions of biomacromolecules Division of Organic Chemistry and Biochemistry Ruđer Bošković Institute, Zagreb, Croatia Isothermal titration calorimetry (ITC) - characterization of binding reactions between proteins and ligands or other macromolecules - enzyme kinetics Differential scanning calorimetry (DSC) - characterization of the thermal stability of proteins and other macromolecular assemblies Isothermal titration calorimetry (ITC) Isothermal titration calorimetry (ITC) VP-ITC PEAQ-ITC ITC – binding ITC – enzyme kinetics • based on simple Michaelis-Menten mechanism kcat - constant that describes the turnover rate of an enzyme-substrate complex to k k ES 1 kcat cat tot E + S ES E + P v product and enzyme k1 Km S KM - Michaelis constant that describes the • Two experimental methods: amount of substrate needed for the enzyme - single injection method (SIM) to obtain half of its maximum rate of reaction - multiple injection method (MIM) ITC – enzyme kinetics - SIM 10 START STOP 9 8 SINGLE CONTINUOUS INJECTION µcal/sec 100 ul 2.2 mM 2'CMP injected in 16 minutes into .06 mM RNase 7 6 0.00 5.00 10.00 15.00 20.00 Time (min) ITC – enzyme kinetics - MIM dP[] 1 dQ v dt Vr H dt dQ/dt cal/sec) µ P( K = 80 M M Rate (millimoles/l/sec) k = 215 s-1 cat Time(sec) [S] (mM) Differential scanning calorimetry (DSC) The Nano DSC Cutaway views of the Nano-DSC New Nano DSC • Platinum capillary cells • New USB connection to computer • Innovative sensor design • Unmatched sensitivity The Nano DSC Cell Geometry . -
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SCIENTIFIC CORRESPONDENCE For these reasons, it is unlikely that the does not affect our analysis of unfolding as chaperones, whereas unfolding does not unfolding approach will provide new in it occurs after the rate-determining step depend on these factors. Far from the sight into the still enigmatic process of in the unfolding direction and accumu unfolding approach being unlikely to protein folding. lates only slightly in the transition region provide new insight into protein folding , it JOHANNES BUCHNER (unpublished data). should be clear from the above that un lnstitut fiir Biophysik und Physikalische Third, we stated clearly that micro folding studies are an essential element in Biochemie, scopic reversibility applies under identical the understanding of folding, and are just Universitat Regensburg, reaction conditions and for a reversible as important as refolding studies. Universitatstrasse 31< process (ref. 1; see also ref. 7, p. R9-90). It A.R. FERSHT D-8400 Regensburg, holds not just for "strongly denaturing J.T. KELLISJR FRG conditions" but for all concentrations of A.T.E.L. ~ATOUSCHEK urea, because folded and unfolded protein l. SERRANO THOMAS KIEFHABER always exist in equilibrium even though MRC Unit for Protein Function and Laboratorium fiir Biochemie, the equilibrium constant may be far from Design, Universitat Bayreuth, unity. At any one of these concentrations, University Chemical Laboratory, Postfach 10 12 51, the transition states for folding and un Lensfield Road, D-8580 Bayreuth, folding are the same. The question is Cambridge CB2 1EW, FRG whether or not the transition state changes UK with change of denaturant. Creighton" FERSHT ET AL.